Class 1

1. BLASTn

Use Nucleotide BLAST and search against the Nucleotide collection (NR database) that includes GenBank.

Here is the input.

Here are the BLASTn top 10 results.

The top hit is Homo sapiens hemoglobin subunit beta (HBB), mRNA.

Here is the information for this sequence (HBB).

Here is more detailed detailed information by clicking the Gene link and redicted to a new page.

2. NCBI

Use NCBI and search for HBB. Select Nucleotide from the drop menu associated with the top search box, and choose RefSeq of Homo sapiens on the left.

Here is the search result (Graphic).

Here is the search result (FASTA).

3. Muscle

Now we have two sequences.

Use Muscle available on the EBI website to align the two sequences above.

Here is the alignment result.

Download the alignment file by clicking Download Alignment File link and redicted to a new page. Then, click File > Save Page As > …. The muscle.clw is saved in the data folder.

4. Seaview

Open downloaded Seaview application. Then, click File > Open > … and select muscle.clw.

Here is what it looks like.

As shown in the picture, there is a Glu6 > Val6 point mutation.

The mutation causing sickle cell anemia is a single nucleotide substitution (A to T) in the codon for amino acid 6. The change converts a glutamic acid codon (GAG) to a valine codon (GTG). Changing a hydrophilic amino acid to a hydrophobic one. Here is more about sickle cell anemia.

5. PDB

We could find sickle cell haemoglobin structures via a text search of main PDB website. However, as we know the nucleotide sequence from our previous work, lets use BLASTX to search the PDB database from the NCBI site.

Here are the BLASTx top 10 results. Notice that the second PDB structure has 100% identity as our example sequence. The sequence ID is 1HBS_B.

6. NGL Viewer

Use online NGL Viewer to visualize the protein structure. NGL Viewer has more advanced display options than the other currently available viewers.

Find 1HBS by clicking File > PDB > …, entering 1HBS and pressing <return>.

Here is what 1NBS looks like.

On the right, click the menu icon next to 1HBS, set Assembly to AU,

and click the menu icon next to 1HBS, set Representation to spacefill,

and set spacefill to 6:H. This will lead to only our mutated amino acid (namely Val 6 in Chain H) being rendered as spacefill.

Here is what the protein looks like.